The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production The GenBank accession numbers for the sequences reported in this paper are AF216700, AF216701 and AF216702.

Woods, Rick; Burger, Michelle; Beven, Carie-Anne; Beacham, Ifor R.

doi:10.1099/00221287-147-2-345

Cited by 83 publications

(69 citation statements)

References 37 publications

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“…The model protein for the RTX family, alpha-hemolysin, inserts into host membranes and forms pores that allow an influx of Ca 2ϩ into host cells, altering host physiology or leading to cell death (37). Additional RTX family members have displayed a wide array of functions in bacterial pathogens; secreted proteases (24,29) and lipases (9,40), cross-linkers of cellular actin that cause host cell rounding (28), and surface-associated coats of protein that form the bacterial S-layer (26,30) represent a few examples of these diverse functions. However, most RTX family members remain uncharacterized.…”

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confidence: 99%

The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

et al. 2012

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Uropathogenic Escherichia coli (UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC. tosA, found in strains within the B2 phylogenetic subgroup of E. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence of tosA in an E. coli isolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function of tosA revealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs. R epeat-in-toxin (RTX) proteins are widespread among Gramnegative bacteria, with more than 1,000 family members detected in a survey of genome sequences from 251 bacterial species (21). Two common features present in known RTX family members are a characteristic glycine-and aspartate-rich repeat near the C terminus of the protein and a conserved type 1 secretion system (T1SS) that exports the protein into the extracellular environment, bypassing the periplasmic space (21, 37). The model protein for the RTX family, alpha-hemolysin, inserts into host membranes and forms pores that allow an influx of Ca 2ϩ into host cells, altering host physiology or leading to cell death (37). Additional RTX family members have displayed a wide array of functions in bacterial pathogens; secreted proteases (24, 29) and lipases (9, 40), cross-linkers of cellular actin that cause host cell rounding (28), and surface-associated coats of protein that form the bacterial S-layer (26, 30) represent a few examples of these diverse functions. However, most RTX family members remain uncharacterized. Given the widespread distribution and diverse roles that known family members contribute to bacterial pathogenesis, the identification and characterization of novel RTX family members remains an important area of research.One of the best characterized RTX family members, alphahemolysin, enhances host damage in the urinary tract during an Escherichia coli infection (32). In addition, this protein contributes to...

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The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

et al. 2012

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“…Protease production by Y. ruckeri (46), Flavobacterium psychrophilum (47) and lipase production by Pseudomonas fluorescens (11,54) has been also demonstrated to be higher at temperatures below the optimal growth temperature. Despite the mechanisms of regulation by temperature are poorly understood, Woods et al (54) suggested that on lipase production by P. fluorescens a posttranscriptional or posttranslational regulation mechanism is involved. In this bacterium, the gene for lipase (lipA) is part of the aprX-lipA operon that includes the genes for a metalloprotease (aprX), for a protease inhibitor and for type I secretion functions.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Two Distinct Metalloproteases Secreted by the Entomopathogenic Bacterium Photorhabdus sp. Strain Az29

Cabral

Cherqui

Pereira

et al. 2004

Appl Environ Microbiol

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Photorhabdus sp. strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures. The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism. The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase. Protease secretion was higher in cultures growing at lower temperatures. At 10°C the activity was highest and remained constant for over 7 days, whereas at 23 and 28°C it showed a steady decrease. Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography. PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50°C. Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14. Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence. In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B. PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA. Its activity ranged between 10 and 80°C and was optimal at pH 7 and 50°C. PrtS also destroyed insect antibacterial factors. Three fragments of PrtS showed homology with a putative metalloprotease of P. luminescens TTO1. Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules.Photorhabdus spp. are non-free-living Enterobacteriaceae (6, 19). The entomopathogen species of Photorhabdus have a symbiotic relation with nematodes of the genus Heterorhabditis for transport with their infective juveniles (IJs). The IJs actively seek for an insect host, penetrating through its natural openings and cuticle. Inside the insect hemocoel, the bacteria are released and actively multiply avoiding the host defenses and causing an acute disease condition that is followed by insect death within 48 h. The bacteria also create the nutritional conditions and protective environment for the development of its nematode symbiont (2, 21).Bacteria of the Photorhabdus genus produce toxins and other potentially virulent factors (16). The characterized toxins are organized in pathogenic islands (53) and include the Tc complex, which is responsible for oral toxicity (9, 52), and the Mcf toxin, which causes loss of insect body turgor, followed by death (14). Among other potential virulence factors, there is a complex set of extracellular enzymes, including proteases, lipases, lecithinases, chitinases, and phosphatases (5,12,20). Proteases represent an important part of these enzymes, although their role on the virulence process is yet unclear. Until now, all extracellular proteases purified and characterized from Photorhabdus were classified as metalloproteases, but in reports on proteases from different strains it has been suggested that ther...

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“…AJ007827). In P. fluorescens B52, Woods et al (2001) reported that expression of the serralysin-type metalloprotease gene, aprX, was negatively regulated by iron at the transcriptional level. While the authors suggested the possible involvement of a PbrA homologue, they were unable to test this hypothesis, as they failed to isolate a pbrA insertion mutant of this strain.…”

Section: Introductionmentioning

confidence: 99%

Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore

2006

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In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

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The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production The GenBank accession numbers for the sequences reported in this paper are AF216700, AF216701 and AF216702.

Cited by 83 publications

References 37 publications

The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

Purification and Characterization of Two Distinct Metalloproteases Secreted by the Entomopathogenic Bacterium Photorhabdus sp. Strain Az29

Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore

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